P630: TARGETING NEGATIVE REGULATION OF MAPK SIGNALING IN CHRONIC LYMPHOCYTIC LEUKEMIA
Authors: M. Stumpf, V. Ecker, P. Giansanti, J. Lu, T. Zenz, I. Ringshausen, B. Küster, J. Ruland, M. Buchner
Journal: HemaSphere (2022)

Background: The MAPK-ERK pathway is a key signaling cascade for cellular homeostasis. Following MAPK activation, feedback loops increase the production of pathway inhibitors, such as dual specificity phosphatases (DUSPs), to restrict MAPK signal. DUSPs can inhibit tumor growth by preventing ERK activation and may act as oncogenes by helping tumor cells in adjusting to elevated MAPK levels. Despite mounting evidence that MAPK signaling is critical for CLL formation and progression, little is known about the role of MAPK negative feedback via the DUSP family in CLL. Aims: In this study, we investigated the significance of DUSP1 and DUSP6’s negative regulation of MAPK in CLL and evaluated their usage as possible therapeutic targets in drug resistant disease. Methods: We examined the expression levels of DUSP1 and DUSP6 in primary CLL patients using RNAseq and immunoblot analysis. To determine their functional relevance, we employed the small molecule inhibitor BCI on primary murine and human (drug resistant) CLL cells in vitro and in vivo and generated genetic knockout MEC-1 CLL cells, to validate the inhibitor effects. In addition, we investigated the downstream effects of DUSP1/6 inhibition on CLL signaling events using global phosphoproteomics in primary CLL and MEC-1 cells. Finally, using particular inhibitors in in vitro experiments, we confirmed the importance of discovered pathway activations. Results: To evaluate a potential role of negative feedback regulation of MAPK signaling in CLL, we first analyzed mRNA expression levels of DUSP1 and DUSP6 in CLL samples (n=210). Both negative regulators were expressed in all of the CLL samples studied, with DUSP6 levels being upregulated in cases with activating mutations in the KRAS or BRAF genes (12.1±17 RNAseq counts for BRAF/KRASwt cases vs. 46.7±19 RNAseq counts for BRAF/KRAS mutated cases). Furthermore, after microenvironmental stimulation, DUSP levels increased in CLL lymph nodes compared to peripheral blood (DUSP1: log2-fold change of 2.6, p=0.0094; DUSP6: log2-fold change of 1.8, p=2.2*10e-7; Herishanu et al, 2011) and stromal coculture. Notably, increased DUSP6 mRNA expression was linked to a poor clinical outcome, as evidenced by a shorter time to treatment (p=0.0109; Log-rank Test) and overall survival (p=0.0075; Log-rank Test). We next used the dual-specific DUSP1/6 inhibitor BCI to block the phosphatase activity and found that this causes cell death in CLL cells, but not or only mildly affected the survival of healthy donor B cells or other T- and B-cell lines. In the TCL1-driven CLL mouse model, administration of the DUSP1/6 inhibitor is effective in lowering CLL cell progression (>50% reduction of total CLL cell count in the spleen; n=5; p=0.0029). We generated genetic knockout lines of DUSP1 or DUSP6, which inhibited CLL cell expansion, to confirm the inhibitor’s specificity. We next used phosphoproteomics to explore the signaling consequences of DUSP1/6 inhibition in CLL, and discovered that DUSP1/6 inhibition caused acute stimulation of BCR/MAPK signaling. Subsequently, the DNA damage response (DDR) pathway is activated, resulting in the induction of apoptotic cell death. Finally, we tested whether DUSP1/6 inhibition is efficacious in drug-resistant CLL by using BCI to treat therapy-naive and ibrutinib refractory CLL samples, and we discovered that DUSP1/6 inhibition is extremely successful in targeting treatment-resistant CLL. Summary/Conclusion: Conclusion: Based on these results, we propose transient DUSP1/6 inhibition as a novel and unexpected concept for eliminating drug-resistant CLL cells.